Hybridoma culture 5E113 was produced after fusion of BALB/c spleen cells from mice immunized with clone 3-5 of LS174T human colon cancer cells and a nonsecretor clone of P3 mouse myeloma cells. Monoclonal antibody (MAB) was evaluated for binding on cell lines and cryostat-sectioned paired human tumorous and normal tissues. No detectable binding was observed for esophageal, lung, kidney, stomach, and breast tumor tissues and paired normal specimens, or to CEA and CSAp. Binding was observed in one of two epiglottal tumor and normal tissues. The antigen recognized by 5E113 MAB appears to recognize an antigen epitope present in glandular structures in some colon tumors and normal bowel tissues. Present efforts are directed toward isolation and characterization of the putative antigenic moiety. The antigen is extractable with 3M KCl or by whole cell lysis, but is not released by 2% 1-butanol treatment. Cell lysis by nitrogen bomb, Polytron or Dounce homogenization in the presence of proteolytic enzyme inhibitors, was followed by ultracentrifugation and membrane dialysis fractionation, yielded 167-, 53-, and 28-fold increases in antigen-specific activity, respectively. The antigen was found in fractions from Mr = 5 to 30 kilodaltons. HPLC and ELISA analyses of lysates from each fraction revealed a family of peaks of antigen activity between Mr = 2.2 and 30 kilodaltons, with Mr = 15 to 17 kilodaltons being the predominant species. Physiochemical studies revealed the antigen not shed into the culture medium. It is inactivated by heating at 85~C for 10 min and by trypsin and pronase treatment, but not by papain, RNAase, DNAase, chymotrypsin, or neuraminidase exposure. The moiety appears to have disulfide bonds. Indirect immunofluorescence on living cells showed pinpoint surface fluorescence, but fixed cells had large amounts of antigen in the cytoplasm. Current studies are directed toward characterization and comparison of this antigen plus a similar antigen recognized by colon tumor monoclonal antibody SW1083-17-1A. Since antigen recognized by 5E113 MAB is found in at least four apparent high molecular weight forms, we are using other isolation procedures to identify a high molecular weight parent molecule. Future studies include continuation of antibody specificity studies and development of anti-idiotype reagents made to the 5E113 and SW1083 17-1-A MABs for use as immunogens for human colon cancer immunity. (AG)